Method for regulating blood glucose level

ABSTRACT

A method for increasing the expression of SIRT1 mRNA and/or decreasing the expression of SOCS3 mRNA, and especially for regulating blood glucose levels in a subject in need thereof is provided. The method comprises administering to the subject an effective amount of an active component selected from the group consisting of a compound of formula (I), a pharmaceutically acceptable salt of the compound of formula (I), and combinations thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein X is H or C1-C3 alkyl; one of Y and Z is 
           
         
       
    
     
       
         
         
             
             
         
       
     
     and the other one is H, OH or 
     
       
         
         
             
             
         
       
     
     wherein when Y is 
     
       
         
         
             
             
         
       
     
     Z is 
     
       
         
         
             
             
         
       
     
     R1 to R13 are independently H or OH, and wherein, R1 to R3 are not simultaneously H; R8 and R9 are not simultaneously H.

This application claims benefit to U.S. Application Ser. No. 61/828,724,filed on May 30, 2013. The entirety of the aforementioned application isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for increasing the expressionof SIRT1 mRNA and/or decreasing the expression of SOCS3 mRNA, andespecially for regulating the blood glucose level in a subject in needthereof and to a medicament for such treatment, and especially relatesto the use of an active component available from an Cistanche tubulosaextract in the treatment of diabetes mellitus.

2. Description of the Related Art

Diabetes mellitus is a chronic metabolic disorder causes by insufficientinsulin secretion or the ineffective use of glucose in the tissue of anorganism, which will leads to excessively high level of blood glucose.It is known that insulin is secreted by pancreatic β cells. Insulin iseffective in regulating the blood sugar and may stimulate the glucosetransport in adipose cells and muscle cells. Obesity, aging and otherreasons may cause insufficient insulin secretion or poor insulinsensitivity, resulting in the increase of blood glucose level anddiabetes mellitus. Patients of diabetes mellitus may suffer fromsymptoms such as, laziness, thirst, excessive urination, blurred vision,and weight loss. Long-term high blood sugar levels may lead to thedysfunction and failure of various organs.

Diabetes mellitus can be primarily classified into Type 1 diabetes andType 2 diabetes. Type 1 diabetes, also known as insulin dependentdiabetes mellitus, is caused by an infection or environmental toxinswhich trigger the patient's own immune system to attack pancreatic βcells. As a result, pancreatic β cells are damaged, resulting inabsolute insulin deficiency and causing blood sugar levels to rise. TypeI diabetes accounts for approximately 5% to 10% of all patients withdiabetes mellitus. Most patients of Type 1 diabetes are diagnosed beforethe age of 30, and thus Type 1 diabetes is also known as juvenilediabetes mellitus.

Type II diabetes is also known as non-insulin dependent diabetesmellitus. Most patients of Type II diabetes are diagnosed after the ageof 40, and thus, it is also known as adult-onset diabetes mellitus. TypeII diabetes accounts for approximately 90% to 95% of all patients withdiabetes mellitus. Type II diabetes is caused by the resistance toinsulin occurring in cells, which gradually decreases the secretion ofinsulin from pancreatic β cells and causes a diabetic metabolicdisorder. Patients of Type II diabetes often simultaneously suffer fromhyperlipidemia, obesity and other symptoms. The risk factors of Type IIdiabetes includes genetic abnormalities, family history of diabetes,age, obesity (especially abdominal obesity), less physical activity,gestational diabetes, and impaired glucose homeostasis, etc.

The prevalence of diabetes mellitus has increased yearly. According tothe World Health Organization in 2008, it is expected that in 2030,there will be over 300 million people with diabetes mellitus globally.Currently, the methods used in the clinical treatments of diabetesmellitus including exercise, diet, and drug treatment. Drug treatmentincludes insulin injection, oral hypoglycemic drugs, such assulfonylurea drugs (sulfonylureas), biguanide drugs (biguanides),α-glucosidase inhibitor, and insulin sensitizers, etc.

The inventors of the present invention found that a compound of formula(I) has an excellent effect on increasing the expression of SIRT1(sirtuin 1) mRNA and/or decreasing the expression of SOCS3 (suppressorof cytokine signaling 3) mRNA, and is effective in lowering bloodglucose, and thus, can be used for regulating the blood glucose level ina subject in need thereof, especially for treating Type I diabetesand/or Type II diabetes in a subject in need thereof:

wherein X is H or C1-C3 alkyl; one of Y and Z is

and the other one is H, OH or

wherein when Y is

Z is

and R1 to R13 are independently H or OH, wherein, R1 to R3 are notsimultaneously H, and R8 and R9 are not simultaneously H. Preferably,the compound of formula (I) is at least one of compound (1) and compound(2) as follows and can be obtained from a plant extract, such as aCistanche tubulosa extract,

Cistanche tubulosa extract belongs to genus Cistanche. The activecomponents of Cistanche tubulosa primarily are phenylethanoidglycosides, including echinacoside, acteoside, and isoacteoside. Aresearch team of Shanghai University of Traditional Chinese Medicine(China) conducted in vitro glucose consumption assay with the use ofliver cells and conducted in vivo drug-induced hypoglycemic efficacytrials with the use of mice having type I diabetes or type II diabetesinduced by different medicaments. The results showed that acteosideobtained from plantago is effective in promoting the consumption ofglucose and can decrease the fasting blood glucose level by enhancingthe serum insulin level (see Chinese patent publication no. CN 102283854A, which is entirely incorporated herein by reference). Therefore, inthe present invention, the active ingredients of Cistanche tubulosaextract were determined by using an animal model of diabetes mellitusand a hepatic glucose consumption test.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide the use of an activecomponent in the manufacture of a medicament, wherein the medicament isfor increasing the expression of SIRT1 mRNA and/or decreasing theexpression of SOCS3 mRNA, and can be used for regulating the bloodglucose level in a subject in need thereof. The active component isselected from the group consisting of a compound of formula (I), apharmaceutically acceptable salt of the compound of formula (I), andcombinations thereof,

wherein X is H or C1-C3 alkyl; one of Y and Z is

and the other one is H, OH or

wherein when Y is

Z is

and R1 to R13 are independently H or OH, wherein, R1 to R3 are notsimultaneously H, and R8 and R9 are not simultaneously H.

Another objective of the present invention is to provide a method forincreasing the expression of SIRT1 mRNA and/or decreasing the expressionof SOCS3 mRNA, or regulating the blood glucose level in a subject inneed thereof, comprising administering to the subject an effectiveamount of an active component selected from the group consisting of acompound of formula (I), a pharmaceutically acceptable salt of thecompound of formula (I), and combinations thereof.

The detailed technology and preferred embodiments implemented for thepresent invention are described in the following paragraphs accompanyingthe appended drawings for people skilled in this field to wellappreciate the features of the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a curve diagram showing the body weight of Sprague-Dawley (SD)rats treated with different conditions;

FIG. 2 is a curve diagram showing the caloric intake of SD rats treatedwith different conditions;

FIG. 3 is a curve diagram showing the plasma glucose concentration of SDrats treated with different conditions;

FIG. 4 is a bar diagram showing the area under the curve of plasmaglucose concentration of SD rats treated with different conditions;

FIG. 5 is a curve diagram showing the plasma nitric oxide (NO)concentration of SD rats treated with different conditions;

FIG. 6 is a curve diagram showing the plasma TNF-α concentration of SDrats treated with different conditions;

FIG. 7 is a curve diagram showing the plasma IL-6 concentration of SDrats treated with different conditions;

FIG. 8 is a curve diagram showing the SIRT1 mRNA expression level in thehypothalamic brain tissue of SD rats treated with different conditions;

FIG. 9 is a curve diagram showing the SOCS3 mRNA expression level in thehypothalamic brain tissue of SD rats treated with different conditions;

FIG. 10 shows a carbohydrate consumption assay carried out by usingliver cells treated with different conditions; and

FIG. 11 shows a carbohydrate consumption assay carried out by usingliver cells treated with different conditions (showing dose dependence).

DETAILED DESCRIPTION OF THE INVENTION

The following will describe some embodiments of the present invention indetail. However, without departing from the spirit of the presentinvention, the present invention may be embodied in various embodimentsand should not be limited to the embodiments described in thespecification. In addition, unless otherwise state herein, theexpressions “a,” “the” or the like recited in the specification of thepresent invention (especially in the claims) should include both thesingular and plural forms. Furthermore, the term “an effective amount”used in this specification refers to the amount of the compound that canat least partially alleviate the condition that is being treated in asuspected subject when administered to the subject. The term “subject”used in this specification refers to a mammalian, including human andnon-human animals. The term “treat” or “treating” includes theprevention of particular diseases and/or disorders, the amelioration ofparticular diseases and/or disorders, and/or the prevention orelimination of the diseases and/or disorder. The term “regulating theblood glucose level in a subject” refers to changing the concentrationof blood glucose in a subject towards a normal value. The unit“mg/kg-body weight” used in this specification means the dosage requiredper kg-body weight.

The present invention provides the use of an active component in themanufacture of a medicament, wherein the medicament is for increasingthe expression of SIRT1 mRNA and/or decreasing the expression of SOCS3mRNA, or regulating the blood glucose level in a subject in needthereof, and the active component is selected from the group consistingof a compound of formula (I), a pharmaceutically acceptable salt of thecompound of formula (I), and combinations thereof,

wherein X is H or C1-C3 alkyl; one of Y and Z is

and the other one is H, OH or

wherein when Y is

Z is

and R1 to R13 are independently H or OH, wherein, R1 to R3 are notsimultaneously H, and R8 and R9 are not simultaneously H. In formula(I), preferably, X is C1-C3 alkyl, such as methyl, ethyl, linear propylor branched propyl; both Y and Z are not H; and/or two of R1 to R3 areOH. X is more preferred to be methyl.

In one preferred embodiment of the present invention, the compound offormula (I) is of structure formula (A) as follows:

wherein Xa is H or C1-C3 alkyl; and R1a to R13a are independently H orOH, wherein R1a to R3a are not simultaneously H, and R8a and R9a are notsimultaneously H.

In formula (A), preferably, Xa is C1-C3 alkyl such as methyl, ethyl,linear propyl or branched propyl; and two of R1a to R3a are OH. Morepreferably, Xa is methyl. Even more preferably, Xa is methyl, two of R1ato R3a are OH, and both R8a and R9a are OH. An embodiment of thecompound of formula (A) is compound (1) (i.e., echinacoside):

In another preferred embodiment of the present invention, the compoundof formula (I) is of structure formula (C):

wherein Xc is H or C1-C3 alkyl; Ye is H, OH or

and R1c to R13c are independently H or OH, wherein, R1c to R3c are notsimultaneously H, and R8c and R9c are not simultaneously H.

In formula (C), preferably, Xc is C1-C3 alkyl such as methyl, ethyl,linear propyl or branched propyl; and two of R1c to R3c are OH. Morepreferably, Xc is methyl. Even more preferably, Xc is methyl, two of R1cto R3c are OH, and both R8c and R9c are OH. An embodiment of thecompound of formula (C) is compound (2) (i.e., isoacteoside):

Therefore, in some embodiments of the present invention, an activecomponent is used in the manufacture of a medicament for increasing theexpression of SIRT1 mRNA and/or decreasing the expression of SOCS3 mRNA,or regulating the blood glucose level in a subject in need thereof,wherein the active component is selected from the group consisting ofcompound (1), a pharmaceutically acceptable salt of compound (1),compound (2), a pharmaceutically acceptable salt of compound (2), andcombinations thereof,

Examples of a pharmaceutically acceptable salt of the above motionedactive components include, but are not limited to, alkali metal salts,such as sodium salt, potassium salt.

According to the present invention, the medicament for increasing theexpression of SIRT1 mRNA and/or decreasing the expression of SOCS3 mRNA,or regulating the blood glucose level in a subject in need thereof canbe manufactured with the use of an active component selected from thegroup consisting of compound (1), compound (2), and combinationsthereof. In these embodiments, the active component can be provided byextracting a plant such as Cistanche tubulosa and thus, the activecomponent can be used as an extract.

A Cistanche tubulosa extract comprising compound (1) and/or compound (2)can be prepared by a method comprising the following steps: (a)extracting Cistanche tubulosa with a polar solvent to provide an extractsolution; and (b) optionally drying the extract solution. The polarsolvent is water and/or a C1-C4 alcohol, such as methanol, ethanol,ethylene glycol, propanol, isopropanol, propylene glycol, n-butanol,isobutanol, t-butanol, butylene glycol or a combination thereof.

The polar solvent is preferred to be selected from the group consistingof water, methanol, ethanol, and combinations thereof. The polar solventis more preferred to be water, ethanol, or a combination thereof. Theamount of the polar solvent and Cistanche tubulosa may be optionallyadjusted. In general, the volume ratio between the polar solvent andCistanche tubulosa may range from about 1:1 to about 50:1, andpreferably about 5:1 to about 20:1.

There is no limitation on the parts of Cistanche tubulosa for used inproviding the Cistanche tubulosa extract. For example, the Cistanchetubulosa extract can be provided from extracting the stem, flower, orthe whole plant of Cistanche tubulosa. According to one embodiment ofthe present invention, the succulent stem of Cistanche tubulosa was usedto provide the extract.

In step (a), the extraction is carried out for a period of time toachieve the desired extraction efficiency. For example, when water isused as the polar solvent, the extraction time is usually at least 15minutes, preferably at least 30 minutes, and more preferably at least 60minutes. Optionally, the extraction may be accompanied with otherappropriate extracting approaches (e.g., stewing, cooling, filtration,concentrated under reduced pressure, and resin column chromatography,etc.) to enhance the efficiency of extraction. Optionally, one mayrepeat the extraction step (a) one or more times with the same ordifferent solvent(s), and combine all the liquid phase thus obtained toprovide the extract solution for step (b) to extract as much activecomponents contained in the plant as possible. Furthermore, one mayrepeat the cycle of step (a) and step (b) for one or more times toremove as much inactive components as possible.

In one embodiment in accordance with the present invention, a Cistanchetubulosa extract was prepared by a method as follows. Cistanche tubulosawas soaked in water, stewed, and filtered, and the cycle was repeatedthree times. The filtrates from different cycles were combined andconcentrated under vacuum to provide a concentrate with a specificgravity of 1.10. Thereafter, ethanol was added into the concentrate to aconcentration of 60%, and then refrigerated for 12 hours. Thesupernatant was collected and concentrated under vacuum to provide acrude extract with a specific gravity of 1.10 and the ethanol wasrecovered. Then, the crude extract was dissolved in hot water with thesame volume as the crude extract to provide a mixture. The mixture wasinjected into a macro-pore absorption resin column for purification. Thecolumn was sequentially eluted with water and ethanol. The ethanoleluent was collected and dried by concentration to provide a Cistanchetubulosa extract. The Cistanche tubulosa extract thus provided comprisesa relatively large amount of compound (1) and a relatively small amountof compound (2).

According to the present invention, the medicament manufactured with theuse of a compound of formula (I), a pharmaceutically acceptable salt ofthe compound of formula (I), or a combination thereof, can be used forincreasing the expression of SIRT1 mRNA and/or decreasing the expressionof SOCS3 mRNA, and especially can be used for regulating the bloodglucose level in a subject in need thereof, such as Type I diabetesmellitus and/or Type II diabetes mellitus. It has been known that SIRT1can promote pancreatic β cells to secrete insulin and can regulate therelated factors that cause insulin resistance, such as free radicals andinflammatory factors, and thereby, improve the conditions caused byinsulin resistance. In addition, it has been known that the expressionlevel of SOCS-3 can be used as an indicator of leptin resistance.Therefore, without being limited by the theory, it is believed that themedicament provided by the present invention can be used for regulatingthe blood glucose level in a subject in need thereof by increasing theexpression of SIRT1 mRNA and/or decreasing the expression of SOCS3 mRNAin the subject, and can be used for treating the diseases related to theexpression levels of SIRT1, such as neuropathy (e.g., Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis (ALS), etc.), cardiovascular diseases(e.g., heart disease, hypotension, hypertension, hyperglycemia, stroke,myocardial infarction, thrombosis, arteriosclerosis, etc.), and obesity.

Depending on the requirements of the subject, the medicament accordingto the present invention can be administered at any suitable dosage. Forexample, when being administered by a human for regulating the bloodglucose level, the medicament is administered at an amount ranging fromabout 0.5 mg to about 100 mg (as the compound of formula (I))/kg-bodyweight per day, and preferably about 1 mg to about 55 mg (as thecompound of formula (I))/kg-body weight per day. However, for patientswith acute conditions, the dosage can be increased to several times orseveral tens of times, depending on the practical requirements.

According to the present invention, the medicament can be in anysuitable form for administration, and be applied in any suitable way.For example, the medicament can be manufactured into a form that issuitable for oral administration, nasal administration, intravenousinjection, subcutaneous injection and/or other methods. Because amedicament in an oral administration form is convenient forself-administration, in one preferred embodiment of the presentinvention, the medicament is provided in an oral administration formsuch as a tablet, a capsule, a granule, powder, a fluid extract, asolution, syrup, a suspension, an emulsion, a tincture, etc. Dependingon the form and purpose, the medicament can further comprise apharmaceutically acceptable carrier.

Using the manufacturing of a medicament suitable for oral administrationas an example, the medicament may comprise a pharmaceutically acceptablecarrier which has no adverse effect on the desired activity of theactive component (i.e., the compound of formula (I) and/or apharmaceutically acceptable salt of the compound of formula (I)), suchas a solvent, oily solvent, diluent, stabilizer, absorption delayingagent, disintegrant, emulsifier, antioxidant, binder, lubricants, andmoisture absorbent. The medicament can be prepared into an oraladministration form by any suitable methods.

As for a medicament suitable for subcutaneous injection or intravenousinjection, the medicament may comprise one or more components such as anisotonic solution, a saline buffer solution (e.g., a phosphate buffer ora citric acid salt buffer), a solubilizer, an emulsifier, and othercarriers to manufacture the composition as an intravenous injection, anemulsion intravenous injection, a powder injection, a suspensioninjection, or a powder-suspension injection.

In addition to the above adjuvants, the medicament may optionallycomprise other additives, such as a flavoring agent, a toner, a coloringagent, etc. to enhance the taste and visual appeal of the resultantmedicament. To improve the storability of the resultant formulation, themedicament may also comprise a suitable amount of a preservative, aconservative, an antiseptic, an anti-fungus reagent, etc. Furthermore,the medicament of the present invention may comprise one or more otheractive components, such as an antioxidant (e.g., vitamin E), insulinsensitizers, etc., to further enhance the efficacy of the medicament orto increase the application flexibility and adaptability of themedicament, as long as the other active components have no adverseeffect on the compound of formula (I) and/or a pharmaceuticallyacceptable salt of the compound of formula (I).

Depending on the requirements of the subject, the medicament accordingto the present invention can be applied with various administrationfrequencies, such as once a day, several times a day, or once for days,etc.

The present invention also provide a method for regulating the bloodglucose level in a subject in need thereof, comprising administering tothe subject an effective amount of an active component selected from thegroup consisting of a compound of formula (I), a pharmaceuticallyacceptable salt of the compound of formula (I), and combinationsthereof. The choice of the active component and its properties, and theformulations and dosages of the active component are all in line withthe above descriptions.

The present invention will be further illustrated in details withspecific examples as follows. However, the following examples areprovided only for illustrating the present invention, and the scope ofthe present invention is not limited thereby.

Example 1 Preparation of Cistanche tubulosa Extract

10 kg succulent stem of Cistanche tubulosa were sliced and soaked ineight times the volume of water for 1 hour, stewed for 2 hours, and thenfiltered. The filtrate was collected. The dregs were mixed with sixtimes the volume of water and stewed twice at 1 hour each time, and thenfiltered. Three obtained filtrates were added together, and thenconcentrated under vacuum at 50° C. to a specific gravity of 1.10.Thereafter, ethanol was added into the concentrate to a concentration of60%, and refrigerated for 12 hours. The clear supernatant liquid wascollected and concentrated under vacuum at 50° C. to provide a crudeextract with a specific gravity of 1.10 and the ethanol was recovered. 6kg of a crude extract were obtained. Then, the crude extract wasdissolved in hot water with the same volume as the crude extract toprovide a mixture. The mixture was injected into a macro-pore absorptionresin column. The column was sequentially eluted with four times thevolume of water and five times the volume of a 40% ethanol. The watereluent was injected in the macro-pore absorption resin column, and theneluted with three times the volume of water. The obtained water eluentwas discarded. The column was then eluted with four times the volume ofa 40% ethanol. The two obtained 40% ethanol eluent were collected anddried by concentration to provide 1107 g of a Cistanche tubulosaextract.

Example 2 Activity Analysis of Cistanche tubulosa Extract

(1) Feeding of the Experimental Animals

4-week-old male SD (Sprague-Dawley) rats were fed for one week, and thenrandomly divided into six groups (10 rats in each group), including fiveexperimental groups and a control group. The rats in the experimentalgroups were intraperitoneally injected with 230 mg/kg body weight ofnicotinamide, and then intraperitoneally injected with 65 mg/kg bodyweight of streptozocin to induced diabetes mellitus (the DM group). Thecontrol group was intraperitoneally injected with the same volume of acitrate buffer (pH 4.5). After a week, the fasting blood glucose of theinduced diabetic rats in the experimental groups were measured, toconfirmed the fasting blood glucose >126 mg/L. The rats were then fedwith a 45% high fat diet (HFD) for 6 weeks. Then, using the dosage shownin Table 1, the rats were orally administered with 0.571 mg/kg bodyweight of rosiglitazone (a medicine for regulating blood glucose levels,which is an insulin sensitizer) (DMR group) or 80 mg/kg body weight ofCistanche tubulosa extract (CTE) (DME1 group), 160 mg/kg body weight ofCTE (DME2 group), or 320 mg/kg body weight of CTE (DME4 group), oncedaily for 6 weeks. The rats in the control group and the DM group wereadministered with double distilled water once daily for 6 weeks. Thebody weight of the rats was measured weekly and the caloric intake wascalculated. The results are shown in FIG. 1 (body weight) and FIG. 2(caloric intake). The results show that there is no significantdifference between the body weight and caloric intake of the rats ineach group.

TABLE 1 Group Experimental conditions and administered compound Controlgroup 45% HFD DM DM + 45% HFD DMR DM + 0.571 mg/kg body weightrosiglitazone + 45% HFD DME1 DM + 80 mg/kg body weight CTE + 45% HFDDME2 DM + 160 mg/kg body weight CTE + 45% HFD DME4 DM + 320 mg/kg bodyweight CTE + 45% HFD

(2) Glucose Tolerance Test

After the rats were treated for 6 weeks as described above, an oralglucose tolerance test (OGTT) was carried out. The rats wereadministered with 2 g/kg body weight of glucose, and then the plasmaglucose concentration was measured after 0, 30, 90, and 120 minutes toanalyze the effect of CTE on the glucose tolerance of the rats afteruptake of glucose. The results are shown in FIG. 3. The Area Under Curve(AUC) of FIG. 3 was calculated and shown in FIG. 4.

As shown in FIGS. 3 and 4, as compared to the DM group, the plasmaglucose concentration in the rats fed with CTE for 6 weeks (the DME1,DME2, and DME4 group) and the rats in the DMR group were decreased at 0,30, 90, and 120 minutes. These results show that CTE can effectivelyincrease the glucose uptake rate of the rats, and thus, can lower bloodsugar levels.

(3) Analysis of Plasma Samples

After the above glucose tolerance test, the rats were sacrificed, andthe blood samples were taken from the celiac artery. The blood sampleswere centrifuged at 3000 rpm, 4° C. for 15 minutes. The supernatant wascollected, which is the plasma. Other tissue of the rats was also takenout and weighed, and then stored at −80° C. for the followingbiochemical data analysis.

(3-1) Determination of Glucose Concentration

20 μl of the plasma was taken and analyzed by a glucose enzymatic kit(FAR EAST GL-V, FAR EAST COMMERCIAL CORP., Japan) to determine thefasting plasma glucose concentration.

The fasting plasma glucose concentration was calculated by the formulaas follows:

Fasting plasma glucose concentration (mg/dl)=(Es−blank)/(Estd−blank)×200

wherein Es: absorbance of the blood sample; blank: absorbance of thesolvent of the kit (without the blood sample); Estd: absorbance of thestandard reagent; 200: concentration of the standard reagent is 200mg/dl. The results are shown in Table 2.

(3-2) Determination of Total Triglyceride Concentration

10 μl of the plasma was taken and analyzed by a tri glyceride enzymatickit (Audit Diagnostics, Cork, Ireland), to determine the totaltriglyceride concentration in the plasma. The total triglycerideconcentration in the plasma was calculated by the formula as follows:

The total triglyceride concentration in the plasma(mg/dl)=(Es−blank)/(Estd−blank)×200

wherein Es: absorbance of the blood sample; blank: absorbance of thesolvent of the kit (without the blood sample); Estd: absorbance of thestandard reagent; 200: concentration of the standard reagent is 200mg/dl. The results are shown in Table 2.

(3-3) Determination of Total Cholesterol Concentration

10 μl of the plasma was taken and analyzed by a cholesterol enzymatickit (Audit Diagnostics, Cork, Ireland), to determine the totalcholesterol concentration in the plasma. The total cholesterolconcentration in the plasma was calculated by the formula as follows:

The total cholesterol concentration in the plasma(mg/dl)=(Es−blank)/(Estd−blank)×200

wherein Es: absorbance of the blood sample; blank: absorbance of thesolvent of the kit (without the blood sample); Estd: absorbance of thestandard reagent; 200: concentration of the standard reagent is 200mg/dl. The results are shown in Table 2.

TABLE 2 mg/dl Group (n = 10 rats/group) (mean ± standard Controldeviation) group DM DMR DME1 DME2 DME4 Fasting blood 82.2 ± 160.82 ±134.37 ± 141.68 ± 129.23 ± 131.04 ± glucose 13.68^(a) 19.71^(c)17.49^(b) 16.41^(b) 18.74^(b) 15.68^(b) Total cholesterol 101.30 ±104.26 ± 106.90 ± 101.85 ± 104.51 ± 81.08 ± 27.92 20.92^(b) 19.26^(b)19.64^(b) 19.78^(b) 11.58^(a) The total 60.82 ± 87.07 ± 62.77 ± 78.46 ±73.44 ± 56.29 ± triglyceride 15.88^(a) 22.42^(b) 16.05^(a) 29.85^(ab)17.88^(ab) 14.74^(a) * One way ANOVA, P < 0.05 as a significance level,post hoc comparisons Dunnett's test after test, wherein “^(a),” “^(b),”and “^(ab)” represent the significant statistical difference between thegroups.

As shown in Table 2, as compared to the DM group, the fasting bloodglucose in the rats fed with CTE for 6 weeks (the DME1, DME2, and DME4group) was decreased. The total triglyceride of the DMR group and DME4group was decreased significantly. It has been known that insufficientinsulin concentration or poor insulin sensitivity in patients ofdiabetes mellitus may cause the abnormal regulation of surroundingadipose tissue, which will result in the decomposition of a large amountof triglycerides in adipose cells. As a result, the concentration offree fatty acids in the blood is increased, resulting in a reducedsensitivity to insulin in peripheral tissues. On the other hand, insulinresistance will lead to the reduction in the use of triglycerides inadipose tissue, resulting in the accumulation of triglycerides in theblood. These results show that CTE is effective in decreasing theconcentration of fasting blood glucose and total triglyceride in thediabetic rats, and thus, can lower blood sugar levels.

(3-4) Determination of Insulin Concentration

25 μl of the plasma was taken and analyzed by an insulin ELISA kit(Mercodia AB Inc., Sylveniusgatan 8A, Sweden). The absorbance wasmeasured at 450 nm wavelength by using an ELISA reader, and theconcentration was calculated based on the standard curve. The resultsare shown in Table 3.

(3-5) Determination of Leptin Concentration

Leptin is a protein hormone secreted by adipose cells. It has been knownthat the imbalance of leptin concentration in the body will causeobesity and the imbalance of insulin. In this experiment, 10 μl of theplasma was taken and analyzed by a leptin enzyme immunoassay kit (AssayDesigns, Ins., Ann Arbor, USA). The absorbance was measured at 450 nmwavelength by using an ELISA reader, and the concentration wascalculated based on the standard curve. The results are shown in Table3.

(3-6) Calculation of the Homeostasis Model Assessment Equation ofInsulin Resistance

The homeostasis model assessment equation of insulin resistance(HOMA-IR) was calculated by the formula as follows:

HOMA-IR=fasting plasma insulin concentration (mU/ml)×fasting plasmaglucose concentration (mmole/L)÷22.5

The results are shown in Table 3.

TABLE 3 ng/ml (mean ± Group (n = 10 the rats/group) standard Controldeviation) group DM DMR DME1 DME2 DME4 Insulin 3.33 ± 5.41 ± 3.57 ± 3.17± 2.34 ± 2.28 ± 1.83^(a) 2.28^(b) 2.15^(a) 1.74^(a) 2.03^(a) 1.43^(a)Leptin 2.97 ± 7.36 ± 6.38 ± 6.26 ± 3.66 ± 3.57 ± 1.51^(a) 1.76^(b)2.05^(b) 2.68^(b) 3.05^(a) 1.71^(a) HOMA-IR 1.38 ± 4.43 ± 2.37 ± 2.45 ±1.62 ± 1.59 ± (μg · 0.70^(a) 1.87^(b) 1.21^(a) 1.79^(a) 1.57^(a)1.04^(a) mmole/L²) *One way ANOVA, P < 0.05 as a significance level,post hoc comparisons Dunnett's test after test, wherein “^(a)” and“^(b)” represent the significant statistical difference between thegroups.

As shown in Table 3, as compared to the control group, the plasmainsulin concentration, plasma leptin concentration, and HOMA-IR in therats of the DM group were decreased. As compared to the DM group, theplasma insulin concentration, plasma leptin concentration, and HOMA-IRin the rats fed with CTE for 6 weeks (the DME1, DME2, and DME4 group)was restored (decreased), and the concentrations shown in DME4 group wasclosed to that of the control group. These results show that CTE canincrease the insulin sensitivity, and decrease leptin resistance andinsulin resistance in the rats.

(3-7) Determination of the Concentrations of Urea, Creatinine, AlanineAminotransferase, and Aspartate Aminotransferase

It has been known that diabetes mellitus may cause a lot ofcomplications. In this experiment, the concentrations of the liver andkidney function indicators in the plasma of the rats, including urea,creatinine, alanine aminotransferase (ALT), and aspartateaminotransferase (AST), were analyzed by an UREA detection kit (UR221,Randox, UK), a creatinine detection kit (CR510, Randox, UK), an ALTdetection kit (AL 1268, Randox, UK), and an AST detection kit (AS 1267,Randox, UK), respectively. The results are shown in Table 4.

TABLE 4 (mean ± Group (n = 10 the rats/group) standard Controldeviation) group DM DMR DME1 DME2 DME4 Urea (mg/dl) 21.30 ± 32.90 ±19.14 ± 23.17 ± 27.14 ± 20.98 ± 4.50^(a) 5.68^(c) 3.92^(a) 6.25^(ab)6.18^(b) 4.21^(a) Creatinine 0.86 ± 2.13 ± 1.19 ± 0.58 ± 0.72 ± 0.66 ±(mg/dl) 0.42^(ab) 0.81^(c) 0.32^(b) 0.20^(a) 0.21^(a) 0.26^(a) AST (U/L)32.36 ± 55.17 ± 38.24 ± 30.90 ± 31.25 ± 34.98 ± 4.65^(a) 23.07^(b)7.03^(a) 7.90^(a) 4.82^(a) 9.41^(a) ALT (U/L) 31.23 ± 31.54 ± 29.88 ±38.82 ± 31.84 ± 30.77 ± 5.74^(a) 7.48^(a) 5.77^(a) 7.29^(a) 9.36^(a)7.68^(a) AST/ALT 1.07 ± 1.76 ± 1.34 ± 0.80 ± 1.06 ± 1.36 ± 0.20^(ab)0.51^(c) 0.38^(b) 0.15^(a) 0.30^(ab) 0.43^(b) *One way ANOVA, P < 0.05as a significance level, post hoc comparisons Dunnett's test after test,wherein “^(a),” “^(b),” “^(ab),” “^(c)” represent the significantstatistical difference between the groups.

As shown in Table 4, as compared to the control group, theconcentrations of urea and creatinine, and the ALT/AST ratio in the ratsof the DM group were increased. As compared to the DM group, the plasmaurea, the concentrations of the plasma urea, creatinine, and ALT/ASTratio in the rats fed with CTE for 6 weeks (the DME1, DME2, and DME4group) was significantly restored (decreased).

(3-8) Determination of the Concentration of Plasma Antioxidant Enzymes

It has been known that both Type I diabetes mellitus and Type IIdiabetes mellitus may cause the decreased expression of antioxidantenzymes in the body, and thus, increases oxidative stress. In thisexperiment, the concentrations of superoxide dismutase (SOD) catalase,and glutathione peroxidase (GPx) in the plasma of the rats were analyzedby an SOD detection kit, a catalase detection kit, and a GPx detectionkit (Eugene-Chen CO., LTD, TW), respectively, to analyze the effect ofCTE on the expression levels of antioxidant enzymes. The results areshown in Table 5.

TABLE 5 U/mg protein Group (n = 10 the rats/group) (mean ± standardControl deviation) group DM DMR DME1 DME2 DME4 Superoxide dismutase 0.60± 0.40 ± 0.65 ± 0.64 ± 0.63 ± 0.55 ± 0.13^(b) 0.05^(a) 0.15^(b) 0.24^(b)0.25^(b) 0.03^(ab) Catalase 81.58 ± 44.07 ± 60.05 ± 68.24 ± 70.53 ±58.29 ± 32.37^(b) 6.79^(a) 9.92^(ab) 27.80^(b) 33.79^(b) 8.50^(ab)Glutathione 1974.36 ± 606.41 ± 811.08 ± 918.66 ± 948.06 ± 1065.95 ±peroxidase 609.87^(c) 498.49^(a) 359.72^(ab) 228.38^(ab) 501.99^(ab)168.89^(b) *One way ANOVA, P < 0.05 as a significance level, post hoccomparisons Dunnett's test after test, wherein “^(a),” “^(b),” “^(ab),”and “^(c)” represent the significant statistical difference between thegroups.

As shown in Table 5, as compared to the control group, the activities ofsuperoxide dismutase, catalase, and glutathione peroxidase in the ratsof the DM group were decreased. As compared to the DM group, theactivities of superoxide dismutase, catalase, and glutathione peroxidasein the rats fed with CTE for 6 weeks (the DME1, DME2, and DME4 group)and the rats in the DMR group were significantly increased. Theseresults show that CTE can increase the expression levels of antioxidantenzymes, and thus, can improve the oxidative stress caused by diabetesmellitus.

(3-9) Determination of Over-Oxidation of Plasma Lipid

It has been known that diabetes mellitus may cause the increasedover-oxidation of plasma lipid. Malondialdehyde (MDA) can be used as anindicator of lipid over-oxidation. In this experiment, the concentrationof plasma malondialdehyde was determined to analyze the effect of CTE onlipid over-oxidation. 0.5 ml of the plasma was taken and evenly mixedwith 1 ml of a reaction reagent (15% w/v % trichloroacetic acid,dissolved in 0.25 N HCl; and 0.375% w/v % thiobarbituric acid, dissolvedin 0.25 N HCl). Then, the mixture was placed in a water bath at 100° C.for 15 minutes, and cooled. 1 ml of n-butanol was added thereinto,vigorously shocked mixed and centrifuged (1500×g, 10 minutes). Thesupernatant was collected, and its absorbance at 532 nm was measured byusing a spectrophotometer, wherein a PBS buffer was used as a blank and5 nM of 1,1,3,3-tetramethoxypropane was used as a standard. Theconcentration of plasma malondialdehyde was calculated by the formula asfollows:

The concentration of plasma malondialdehyde (nM/ml)=[(absorbance of thesample−blank)/(absorbance of the standard−blank)]×5

The results are shown in Table 6.

TABLE 6 nM/ml (mean ± Group (n = 10 the rats/group) standard Controldeviation) group DM DMR DME1 DME2 DME4 Malondial- 10.15 ± 16.87 ± 13.38± 9.90 ± 10.66 ± 9.95 ± dehyde 1.20^(a) 2.42^(c) 2.12^(b) 2.09^(a)1.94^(a) 1.55^(a) *One way ANOVA, P < 0.05 as a significance level, posthoc comparisons Dunnett's test after test, wherein “^(a),” “^(b),” and“^(c)” represent the significant statistical difference between thegroups.

As shown in Table 6, as compared to the control group, the concentrationof plasma malondialdehyde in the rats of the DM group was increased. Ascompared to the DM group, the concentration of plasma malondialdehyde inthe rats fed with CTE for 6 weeks (the DME1, DME2, and DME4 group) wasdecreased. These results show that CTE is effective in decreasing thelipid over-oxidation caused by diabetes mellitus.

(3-10) Determination of Plasma Inflammatory Indicators

It has been known that the concentration of Advanced GlycationEnd-products (AGE) in diabetic patients will increase. When free AGEscombine with receptor for advanced glycation end products (RAGE),superoxide radicals will be generated, which will activate downstreamNF-κB transcription factors, and thus, result in a series ofinflammatory reactions. In this experiment, the concentrations ofinflammatory indicators in the plasma, including nitric oxide (NO),TNF-α, and IL-6, were determined to analyze the effect of CTE oninflammation caused by diabetes mellitus. 100 ml of standard solutionsof different concentrations or the plasma were added into differentwells of a microplate coated with a capture antibody. The microplate wasincubated at room temperature for 2 hours, the sample was removed, andthen the microplate was washed five times with a 400 μl of a washingbuffer (1×PBS, 0.05% Tween 20). 100 μl of the detecting antibodies ofNO, TNF-α, and IL-6 were added into the microplate and incubated at roomtemperature for 1 hour. Then, the supernatant was discarded, and themicroplate was washed with the washing buffer 5 times. 480 μl of enzymeavidan-HRP was added into each well of the microplate and incubated atroom temperature for 30 minutes. Then, the supernatant was discarded,and the microplate was washed with the washing buffer 5 times. 100 μl ofa substrate solution was added into each well of the microplate andincubated at room temperature for 15 minutes. 50 μl of a stop solutionwas added into each well of the microplate, and the absorbance wasmeasured at 450 nm wavelength by using a microplate reader to calculatethe concentrations. The results are shown in FIGS. 5 to 7. In FIGS. 5 to7, post hoc comparisons was used, wherein “a,” “b,” and “c” representthe significant statistical difference between the groups.

As shown in FIGS. 5 to 7, as compared to the control group, the plasmaconcentrations of NO (FIG. 5), TNF-α (FIG. 6), and IL-6 (FIG. 7) in therats of the DM group was increased, indicating a serious inflammation.As compared to the DM group, the concentrations of NO, TNF-α, and IL-6in the rats fed with CTE for 6 weeks (the DME1, DME2, and DME4 group)were decreased. These results show that CTE is effective in decreasingthe inflammation caused by diabetes mellitus.

(4) Determination of the Weights of the Liver, Kidney, Heart, andAbdominal Fat of the Rats

In this experiment, the weights of the liver, kidney, heart, andabdominal fat of the rats treated with different conditions for 6 weekswere determined. As shown in Table 7, there are no significantdifferences between the weights of the liver, kidney, heart, andabdominal fat of the rats in different groups.

TABLE 7 Percentage (%) compared to body Group (n = 10 the rats/group)weight (mean ± Control standard deviation) group DM DMR DME1 DME2 DME4Liver 3.27 ± 3.51 ± 3.21 ± 3.38 ± 3.22 ± 3.15 ± 0.29^(a) 0.42^(a)0.31^(a) 0.41^(a) 0.40^(a) 0.23^(a) Kidney 0.38 ± 0.34 ± 0.34 ± 0.36 ±0.34 ± 0.36 ± 0.03^(a) 0.04^(a) 0.04^(a) 0.07^(a) 0.04^(a) 0.02^(a)Heart 0.33 ± 0.31 ± 0.34 ± 0.33 ± 0.34 ± 0.33 ± 0.03^(a) 0.02^(a)0.03^(a) 0.04^(a) 0.04^(a) 0.02^(a) Abdominal fat 1.45 ± 2.55 ± 2.49 ±2.30 ± 2.59 ± 1.98 ± 0.33^(a) 0.75^(b) 0.65^(b) 0.57^(b) 0.72^(b)0.50^(ab) *One way ANOVA, P < 0.05 as a significance level, post hoccomparisons Dunnett's test after test, wherein “^(a),” “^(b),” and“^(ab)” represent the significant statistical difference between thegroups.

(5) Determination of the Expression of SIRT1 and SOCS-3

It has been known that the sirtuin 1 (SIRT1) can promote pancreatic βcells to secrete insulin and regulate the related factors that causeinsulin resistance, such as free radicals and inflammatory factors, andthereby, improving the insulin resistance. In addition, the expressionlevel of SOCS-3 (suppressor of cytokine signaling 3) can be used as anindicator of leptin resistance. In this experiment, the effect of CTE onthe expression levels of SIRT1 mRNA and SOCS-3 mRNA of the rats werediscussed. The total RNA of the hypothalamic brain tissue of the ratswas extracted by using an RNeasy Lipid Tissue Mini (QIAGEN). 1 μg of thetotal RNA, 1 μl of 0.5 μg/μl oligo(dT), and DEPC-H₂O (to a final volumeof 12 μl) were mixed. Then, the mixture were reacted at 65° C. for 5minutes, and placed on ice for 5 minutes. 4 μl of a 5× first-strandbuffer, 1 μl of 10 mM dNTP, and 1 μl HiScript I reverse transcriptasewere added thereinto, and then the mixture were reacted at 30° C. for 10minutes, at 48° C. for 60 minutes, and at 70° C. for 15 minutes. Then, 1μl of the obtained DNA, 0.5 μl 10 mM dNTPs, 2.5 μl 10×PCR buffer, 0.5 μlof each gene specific primer (GSP), 0.25 μl Taq polymerase, and doubledistilled water (to a final volume of 25 μl) were mixed, and apolymerase chain reaction (PCR) was conducted at the conditions asfollows: denaturation at 94° C. for 5 minutes followed by 40 cycles ofdenaturation at 94° C. for 30 seconds; annealing at 55° C. for 30seconds; elongation at 72° C. for 30 seconds. The results are shown inFIGS. 8 and 9.

As shown in FIG. 8, as compared to the control group, the SIRT1 mRNAexpression level of the diabetic rats (the DM group) is significantlydecreased. These results show that CTE can increase the expression levelof SIRT1 mRNA of the rats, and thereby, improving insulin resistance. Inaddition, as shown in FIG. 9, as compared to the control group, theSOCS-3 mRNA expression levels of the rats in the DM group aresignificantly increased, indicating more severe leptin resistance. Ascompared to the DM group, the SOCS-3 mRNA expression levels of the ratsfed with CTE (DME1, DME2, and DME4 group) were significantly decreased.These results show that CTE can decrease the expression level of SOCS-3mRNA of the rats, and thereby, improving leptin resistance caused bydiabetes mellitus.

Example 3 Analysis of the Components in Cistanche tubulosa Extract

The components in the Cistanche tubulosa extract were analyzed by highperformance liquid chromatography (HPLC). The experimental conditionswere as follows: using an Agilent Zorbax SB-C18 column (2.1×150 mm, 5μm); the mobile phase was solvent A: acetonitrile (ACN) containing 0.1%formic acid, and solvent B: MQ-H₂O containing 0.1% formic acid; a flowrate of 0.3 ml/minutes; detection wavelength was 333 nm. 5 μl of thestandards of echinacoside (ChromaDex, US), acteoside (ChromaDex, US),and isoacteoside (ChromaDex, US), and the Cistanche tubulosa extractsolution (dissolved in 50% methanol) was separately injected into theHPLC system. The peak areas of each sample were determined, and thepercentage of echinacoside, acteoside and isoacteoside contained in theCistanche tubulosa extract was calculated based on the peak areas. Theresults are shown in Table 8.

TABLE 8 Component Echinacoside Acteoside Isoacteoside Percentage 26.2 wt% 2.6 wt % 4.4 wt %

As shown in Table 8, the Cistanche tubulosa extract comprises about 26.2wt % of echinacoside, about 2.6 wt % of acteoside, and about 4.4 wt % ofisoacteoside.

Based on the data shown in Table 8, the contents of each activecomponent contained in the CTE administered in the DME1, DME2, DME4group described in Example 2 are calculated, and the results are shownin Table 9.

TABLE 9 Dosage (mg/kg) Echinacoside Acteoside Isoacteoside  80 (DME1group) 20.96 2.08 3.52 160 (DME2 group) 41.92 4.16 7.04 320 (DME4 group)83.84 8.32 14.08

Example 4 Analysis of the Activities of Echinacoside and Isoacteoside

The above experimental data show that the Cistanche tubulosa extractcomprises echinacoside, acteoside and isoacteoside. In this experiment,the effect of echinacoside, acteoside and isoacteoside on regulatingblood glucose levels was further confirmed.

(1) Cell Assay

A carbohydrate consumption assay was conducted by using humanhepatocellular liver carcinoma cell line (HepG2, Bioresource Collectionand Research Center, BCRC number: 60025). The cells were treated with1000 μg/ml glucose, and then treated with 100 nM insulin, 50 μg/mlCistanche tubulosa extract (CTE), 12.5 μg/ml of echinacoside, 1.25 μg/mlof acteoside, or 2.25 μg/ml of isoacteoside (i.e., an active componentby an amount of what it presents in the extract). After 1 hour, thecarbohydrate consumption level was determined. The results are shown inFIG. 10, wherein the data were shown as mean±standard deviation (n=3);***P<0.001 (as compared to the control group).

As shown in FIG. 10, as compared to the control group, 50 μg/mlCistanche tubulosa extract and 12.5 μg/ml of echinacoside and 2.25 μg/mlof isoacteoside can effectively increase the carbohydrate consumptionlevel of the liver cells, while the efficacy of 1.25 μg/ml of acteosidewas not obvious, which might be due to its low percentage.

(2) Concentration-Dependent Assay

To further confirm if the active components that are present inCistanche tubulosa extract have the effect of accelerating liver cellsto uptake blood sugar, a carbohydrate consumption assay as describedabove was conducted by using different concentrations of Cistanchetubulosa extract, echinacoside, acteoside and isoacteoside to treatedthe HepG2 cells. The results are shown in FIG. 11, wherein the data wereshown as mean±standard deviation (n=3); **P<0.01; ***P<0.001 (ascompared to the control group); #P<0.05 (as compared to the insulingroup).

As shown in FIG. 11, as compared to the control group, 5 μg/ml, and 50μg/ml of Cistanche tubulosa extract (CTE), echinacoside, acteoside andisoacteoside can effectively increase the carbohydrate consumption levelof the liver cells, and the results showed a dose dependence, whereinthe result of acteoside is similar to previous researches, showing thatacteoside can increase the carbohydrate consumption level of livercells. Furthermore, the results of this experiment show thatechinacoside and isoacteoside can increase the carbohydrate consumptionlevel of liver cells at a very low concentration, such as 5 μg/ml. Theresults of this experiment also show that the effect of Cistanchetubulosa extract in increasing the carbohydrate consumption level ofliver cells and thus in lowering blood sugar levels is primarilyattributable to echinacoside and acteoside contained therein, but notacteoside.

The above results show that Cistanche tubulosa extract and theechinacoside and isoacteoside contained therein are effective inlowering blood sugar levels, and thus, can be used for regulating bloodglucose levels, especially for treating Type I and Type II diabetesmellitus.

What is claimed is:
 1. A method for regulating blood glucose level in asubject in need thereof, comprising administering to the subject aneffective amount of an active component selected from the groupconsisting of a compound of formula (I), a pharmaceutically acceptablesalt of the compound of formula (I), and combinations thereof:

wherein X is H or C1-C3 alkyl; one of Y and Z is

and the other one is H, OH or

wherein when Y is

Z is

and R1 to R13 are independently H or OH, wherein, R1 to R3 are notsimultaneously H, and R8 and R9 are not simultaneously H.
 2. The methodas claimed in claim 1, wherein two of R1, R2 and R3 are OH.
 3. Themethod as claimed in claim 1, wherein X is C1-C3 alkyl.
 4. The method asclaimed in claim 1, wherein the compound of formula (I) is of structureformula (A):

wherein Xa is H or C1-C3 alkyl; and R1a to R13a are independently H orOH, wherein R1a to R3a are not simultaneously H, and R8a and R9a are notsimultaneously H.
 5. The method as claimed in claim 4, wherein Xa isC1-C3 alkyl, and two of R1a, R2a and R3a are OH.
 6. The method asclaimed in claim 4, wherein the compound of formula (I) is compound (1):


7. The method as claimed in claim 1, wherein the compound of formula (I)is of structure formula (C):

wherein Xc is H or C1-C3 alkyl; Yc is H, OH or

and R1c to R13c are independently H or OH, wherein, R1c to R3c are notsimultaneously H, and R8c and R9c are not simultaneously H.
 8. Themethod as claimed in claim 7, wherein Xc is C1-C3 alkyl, and two of R1cto R3c are OH.
 9. The method as claimed in claim 7, wherein the compoundof formula (1) is compound (2):


10. The method as claimed in claim 1, wherein the active component isselected from the group consisting of compound (1), a pharmaceuticallyacceptable salt of compound (1), compound (2), a pharmaceuticallyacceptable salt of compound (2), and combinations thereof:

component is selected from the group consisting of compound (1),compound (2), and combinations thereof.
 12. The method as claimed inclaim 11, wherein the active component is used as an extract.
 13. Themethod as claimed in claim 12, wherein the extract is a Cistanchetubulosa extract.
 14. The method as claimed in claim 13, wherein theCistanche tubulosa extract is prepared by a method comprising: (a)extracting Cistanche tubulosa with a polar solvent to provide an extractsolution; and (b) optionally drying the extract solution, wherein thepolar solvent is selected from the group consisting of water, C1-C4alcohols, and combinations thereof.
 15. The method as claimed in claim14, wherein the polar solvent is selected from the group consisting of:water, methanol, ethanol, and combinations thereof.
 16. The method asclaimed in claim 1 for treating Type I diabetes mellitus.
 17. The methodas claimed in claim 1 for treating Type II diabetes mellitus.
 18. Themethod as claimed in claim 1, wherein the active component isadministered at an amount ranging from about 0.5 mg (as the compound offormula (I))/kg-body weight to about 100 mg (as the compound of formula(I))/kg-body weight per day.
 19. The method as claimed in claim 1,wherein the active component is administered at an amount ranging fromabout 1 mg (as the compound of formula (I))/kg-body weight to about 55mg (as the compound of formula (I))/kg-body weight per day.
 20. A methodfor increasing the expression of SIRT1 mRNA and/or decreasing theexpression of SOCS3 mRNA in a subject in need thereof, comprisingadministering to the subject an effective amount of an active componentselected from the group consisting of a compound of formula (I), apharmaceutically acceptable salt of the compound of formula (I), andcombinations thereof:

wherein X is H or C1-C3 alkyl; one of Y and Z is

and the other one is H, OH or

wherein when Y is

Z is

and R1 to R13 are independently H or OH, wherein, R1 to R3 are notsimultaneously H, and R8 and R9 are not simultaneously H.